Catalytic sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Their number and their kinetic and spectral properties

Peczon, Benigno D.; Spivey, H. Olin

doi:10.1021/bi00762a001

Cited by 30 publications

(12 citation statements)

References 27 publications

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“…The apo-enzyme of GAPDH was prepared using ion-exchange chromatography on CMSepharose (Sigma) as described. 35,36 The protein was desalted using a Sephadex G-25 (Sigma) column (0.8 cm!0.8 cm!35 cm) and concentrated. The concentration of the apo-enzyme was determined by measuring the absorbance at 280 nm using a Cary spectrometer: [E t ] (mM tetramer)ZA 280 /116!dilution (A 280 1.16!10 5 for the apo-enzyme 37 ).…”

Section: Methodsmentioning

confidence: 99%

Ligand Binding Energy and Enzyme Efficiency from Reductions in Protein Dynamics

Williams

Zhou

Stephens

2006

Journal of Molecular Biology

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Section: Methodsmentioning

confidence: 99%

Ligand Binding Energy and Enzyme Efficiency from Reductions in Protein Dynamics

Williams

Zhou

Stephens

2006

Journal of Molecular Biology

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“…NAD', the co-substrate and modulator of GraP-DH enzymic activity [21], was shown to efficiently elute the enzyme at The cytosolic fraction prepared from 2X 10' R B I 3 cells (10 ml) was passed through an ethanolamine-treated CNBr-activated Sepharose column (3 ml). The eluted fraction was loaded onto a C27-ICAM-1 -Sepharose matrix (3 ml) equilibrated with buffer A.…”

Section: Resultsmentioning

confidence: 99%

Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐Tubulin

Federici

Camoin

Hattab

et al. 1996

European Journal of Biochemistry

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To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule-I (ICAM-1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27-aminoacid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM-1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM-1-interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein P-tubulin and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraP-DH). Experiments carried out with purified GraP-DH or CNBr fragments of GraP-DH indicated that binding to the ICAM-1 matrix was mediated by the C-terminal domain of GraP-DH, containing the binding site of the cofactor NAD+, and that NAD' could compete with this binding. Using a series of ICAM-1 C-terminal truncated peptides, we could demonstrate that (a) the nitric-oxideinduced covalent linkage of NAD' to GraP-DH was impaired by these peptides, (b) the glycolytic activity of GraP-DH was drastically inhibited by a truncated peptide containing the 15 C-terminal residues, (c) nitric oxide appeared to prevent this inhibition.Together, our results demonstrate that GraP-DH specifically associate! with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.

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“…It is well known that dissociation of the weakly bound NAD molecules from GAPD is followed by conformational change [13,14] and partial inactivation [6,8] of the enzyme. The general conclusion which can be drawn from the results presented here is that the binding of NAD coenzyme to the phospholipid membranes may be responsible for partial inactivation of GAPD.…”

Section: Resultsmentioning

confidence: 99%

Effect of NAD coenzyme on the inactivation of glyceraldehyde‐3‐phosphate dehydrogenase by anionic phospholipids

et al. 1990

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The inactivation of bovine heart glyceraldehyde-3-phosphate dehydrogenase by phosphatidylinositol (PI) and phosphatidylserine (PS) in the form of liposomes was investigated in the presence and absence of NAD excess. In the absence of NAD, the enzyme activity decreased to about ~0% of its initial value at 0.6 mM PI and 0.8 mM PS (lipid-to-protein molar ratio 600 and 800, respectively). In the same lipid concentration range almost full regainment of the activity was observed in the presence of 80/~M NAD. It was shown that the excess of NAD protects the enzyme against conformational change induced by the phospholipids. Centrifugation experiments showed that both PI and PS bind significant amounts of NAD.Glyceraldehyde-3-phosphate dehydrogenase; Phosphatidylinositol; Phosphatidylserine; Lipid-protein interaction

show abstract

Catalytic sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Their number and their kinetic and spectral properties

Cited by 30 publications

References 27 publications

Ligand Binding Energy and Enzyme Efficiency from Reductions in Protein Dynamics

Ligand Binding Energy and Enzyme Efficiency from Reductions in Protein Dynamics

Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐Tubulin

Effect of NAD coenzyme on the inactivation of glyceraldehyde‐3‐phosphate dehydrogenase by anionic phospholipids

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